Genetic Variation in Phosphodiesterase (PDE) 7B in Chronic Lymphocytic Leukemia: Overview of Genetic Variants of Cyclic Nucleotide Pdes in Human Disease

ORIGINAL ARTICLE

Genetic variation in phosphodiesterase (PDE) 7B in chronic lymphocytic leukemia: overview of genetic variants of cyclic nucleotide PDEs in human disease

Ana M Peiro´ 1,2, Chih-Min Tang1, Fiona Murray1,3, Lingzhi Zhang1, Loren M Brown1, Daisy Chou1, Laura Rassenti4, Thomas A Kipps3,4 and Paul A Insel1,3,4

Expression of cyclic adenosine monophosphate-speciﬁc phosphodiesterase 7B (PDE7B) mRNA is increased in patients with chronic lymphocytic leukemia (CLL), thus suggesting that variation may occur in the PDE7B gene in CLL. As genetic variation in other PDE family members has been shown to associate with numerous clinical disorders (reviewed in this manuscript), we sought to identify single-nucleotide polymorphisms (SNPs) in the PDE7B gene promoter and coding region of 93 control subjects and 154 CLL patients. We found that the PDE7B gene has a 5¢ non-coding region SNP À347C4T that occurs with similar frequency in CLL patients (1.9%) and controls (2.7%). Tested in vitro, À347C4T has less promoter activity than a wild-type construct. The low frequency of this 5¢ untranslated region variant indicates that it does not explain the higher PDE7B expression in patients with CLL but it has the potential to inﬂuence other settings that involve a role for PDE7B. Journal of Human Genetics (2011) 56, 676–681; doi:10.1038/jhg.2011.80; published online 28 July 2011

Keywords: cAMP; chronic lymphocytic leukemia; cyclic nucleotide phosphodiesterases; PDE7B single-nucleotide polymorphisms

INTRODUCTION 410-fold in peripheral blood mononuclear cells of patients with Cyclic nucleotide phosphodiesterases (PDEs), a superfamily of CLL.16 We thus set out to determine if the increased expression of enzymes divided into 11 families (PDE1–11), several with multiple PDE7B in CLL patients might result from genetic variation in the isoforms,1,2 hydrolyze cyclic adenosine monophosphate (cAMP) and coding sequence or 5¢ untranslated region (5¢ UTR) of the PDE7B cyclic guanosine monophosphate (cGMP), and thereby have a crucial gene. We began these studies with the knowledge that genetic varia- role in termination of cyclic nucleotide-mediated signaling, in parti- tion, in particular single-nucleotide polymorphisms (SNPs), had been cular signaling by drugs that regulate the generation of cAMP and identified in certain PDE family members, and suggested to contribute cGMP.3 In addition, PDEs, including specific PDE isoforms, have been to clinical disorders, such as schizophrenia (for example, PDE4B),17–19 used as drug targets. Several PDEs, including PDE4, PDE7 and PDE8, stroke (for example, PDE4D)20 and retinitis pigmentosa (for example, preferentially hydrolyze cAMP.4 In spite of their importance in the PDE6A and PDE6B),21 but evidence regarding such genetic variation regulation of cyclic nucleotides, the mechanisms that regulate the for other PDEs is limited. We thus set out to identify genetic variants expression of PDE genes are not well defined. For example, regions in in the PDE7B gene and to test the hypothesis that genetic variation the promoter that regulate activity have only been identified for a might contribute to the increased PDE7B mRNA expression in CLL limited number of PDE genes5 and details regarding events involved in and to the pathophysiology of CLL with implications for the possible PDE gene expression are not well understood. Increases in cAMP can use of inhibitors of PDE7B in the treatment of CLL. By sequencing the regulate the expression of certain PDEs, in particular via action of the 5¢ UTR of the PDE7B gene, we identified a variant that decreases cAMP-response element-binding protein or the inducible cAMP early promoter activity. In addition, in this article, we place our findings for repressor,6–9 and in addition, glucocorticoids can transcriptionally PDE7B in the context of genetic variation of other human PDEs. inhibit PDE expression.10,11 Our interest in PDE gene expression arose from studies of patients MATERIALS AND METHODS with chronic lymphocytic leukemia (CLL), the most common Patient selection and sample preparation 12 leukemia in North American and European adults. We found that We evaluated samples from 154 CLL patients followed by the CLL Research mRNA and protein expression of PDE7B, a cAMP-specific PDE Consortium and 93 healthy donors at the University of California, San Diego. predominantly expressed in brain and lymphocytes,13–15 are increased Following informed consent, blood was collected from the healthy donors and

1Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA; 2Department of Clinical Pharmacology, Hospital General Universitario de Alicante, Alicante, Spain; 3Department of Medicine, University of California, San Diego, La Jolla, CA, USA and 4Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA Correspondence: Dr PA Insel, Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0636,USA. E-mail: [email protected] Received 29 April 2011; revised 16 June 2011; accepted 17 June 2011; published online 28 July 2011 Single-nucleotide polymorphism in the PDE7B promoter AM Peiro´ et al 677

Table 1 Demographic data of CLL patients and control subjects who have WT PDE7B or those with the À347C4TSNP

Control CLL

SNP (n¼4) WT (n¼89) Total (n¼93) SNP (n¼6) WT (n¼148) Total (n¼154)

Male:female 4:0 73:16 77:16 4:2 92:56 96:58 Age (years) 42±855±13 55±13 64±12 62±10 62±10 IgVH (%) — — — 95±495±996±4 ZAP-70 level (%) — — — 12±18 24±28 23±27 WBC (cells mlÀ1 Â1000) — — — 105±50 87±121 88±119

Abbreviations: CLL, chronic lymphocytic leukemia; IgVH, immunoglobulin heavy chain variable; PDE7B, phosphodiesterase 7B; SNP, single-nucleotide polymorphism; WBC, white blood cell; WT, wild type; ZAP-70, 70-kDa zeta-associated protein. Data for age, IgVH%, Zap70 and WBC are shown as mean±s.d.

from patients who satisﬁed diagnostic and immunophenotypic criteria for CLL. Table 2 Gender-allele frequency analysis of expression of the The University of California, San Diego, Institutional review board approved À347C4T variant of PDE7B these studies and the CLL Research Consortium approved the procurement of the samples, with all approvals in accordance with the Declaration of Helsinki. Gender Number Alleles SNP/total alleles Diagnosis of CLL was based on morphological and immunophenotyping criteria.22 Patients with CLL were also categorized by the expression of 70-kDa Male zeta-associated protein.23–25 Mononuclear cells were isolated using a Ficoll CLL 96 4 4/192 (2%) gradient and then frozen in fetal calf serum plus 10% dimethyl sulfoxide before Control 77 5 5/154 (3.3%) storage in liquid N2. The 70-kDa zeta-associated protein expression was deter- Total 173 9 9/346 (2.6%)* mined by ﬂow cytometry of mononuclear cells. PCR was used to assess the immunoglobulin heavy chain variable gene family. Table 1 shows demographic Female data, heavy chain variable and the 70-kDa zeta-associated protein status. CLL 58 2 2/116 (1.7%) Control 16 0 0/32 (0%) Genomic DNA and RNA extraction, PCR and sequencing Total 74 2 2/148 (1.4%)*

Genomic DNA and RNA were prepared using isolation kits for blood cells Abbreviations: CLL, chronic lymphocytic leukemia; PDE7B, phosphodiesterase 7B; SNP, single- (Qiagen, Valencia, CA, USA). The complementary DNA (cDNA) was generated nucleotide polymorphism. using the Superscript III cDNA synthesis system (Invitrogen, Carlsbad, CA, *P-values were 40.1 for differences in expression of the variant between control males and males with CLL and between males and females. USA) according to the manufacturer’s instructions. We sought to identify genetic variants within the putative PDE7B (NC_000006.11) promoter and coding regions as follows: (a) PDE7B gene promoter, the region 700 bp * upstream and 300 bp downstream of the transcription start site of PDE7B 8 ** was amplified; this resulted in a 1-kb PCR product (P1; forward primer: 5¢-CACCATAGCCTTGCTTCTTGA-3¢; reverse primer, 5¢-TGGCAGTTCTGTG 7 ACCTTTG-3¢). (b) PDE7B coding region (304 to 1656 nt), the full-length 6 ** cDNA of PDE7B was amplified in two PCR products, P2 from 276 to 1165 bp 5 (forward primer: 5¢-CTGGAGAAGTTGCTGGATTCT-3¢; reverse primer: 5¢-ATT 4 CATTCTGCCTGTTGATGTCT-3¢) and P3 from 1074 to 1870 bp (forward primer: 5¢-CTCTGGACATCATGCTTGGA-3¢; reverse primer: 5¢-CTCCCACG 3 TTACTGAATGGAG-3¢). PCR amplifications were performed (AccuPower PCR 2 Premix; Bioneer, Alameda, CA, USA) using standard procedures with 50 ng of Activity Luciferase 1 genomic DNA product (P1) or 200 ng of cDNA (P2–3), in a total volume of vector) increase over (fold 20 mLcontaining10mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl ,1U 0 2 vec WT SNP DNA polymerase, 1.5 mM deoxyribonucleotide triphosphate and 0.5 mM each specific primer. PCR cycling conditions were: initial denaturing at 95 1Cfor Figure 1 Impact of the À347C and À347T genotypes on PDE7B promoter 5 min (P1) or 2 min (P2–3) followed by 35 cycles of denaturing at 94 1Cfor function. pGL4 luciferase reporter constructs containing a 1-kb phospho- 40 s; annealing at 59 1C(P1),551C(P2)or571C (P3) for 40 s; extension at diesterase 7B promoter with the À347C (WT) or À347T (SNP) genotype 72 1C for 57 s (P1), 59 s (P2) or 58 s (P3); and a final extension step at 72 1Cfor were transfected into HEK293 cells. Vec, the promoter-less pGL4, was used 5 min. DNA Clean & Concentrator TM-5 kit (D4003 lt 0940 BW) and Centri- as a control and its luciferase activity was defined as 1. The data represent sep columns (Zymo Research, Orange, CA, USA) were used to purify PCR the mean±s.d. of three independent experiments. **Po0.001 compared products. Sequence was determined on a 3130Âl Genetic Analyzer (Applied with vec alone and *Po0.001 between WT and SNP. SNP, single-nucleotide polymorphism; vec, vector; WT, wild type. Biosytems, Carlsbad, CA, USA), with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Polymorphisms were manually confirmed. The data were validated visually and the files were inspected to identify that contained either wild type (À347C) or SNP (À347T). The 1-kb fragment heterozygotes. Each sequence was imported into the Biology Workbench encompassing the PDE7B transcription start site (from À700 to +300) was (http://workbench.sdsc.edu/) for analysis. SNPs were confirmed by re-sequen- amplified from patients with the À347 CC or À347 TT genotype, and inserted cing from the reverse direction. into the firefly luciferase reporter plasmid pGL4-basic (Promega, Madison, WI, USA). HEK293 cells were transfected with 3 mg of pGL4-Luc containing PDE7B Dual luciferase assays promoter regions of either the wild-type or SNP variants and 0.1 mgofpRL- To investigate the functional impact of À347 C4T polymorphism on PDE7B TK-Luc using the Optifect transfection reagent (Invitrogen, Carlsbad, CA, promoter activity, we constructed reporter plasmids with 5¢-flanking regions USA). pRL-TK-Luc encodes the Renilla luciferase under the control of an

Journal of Human Genetics Single-nucleotide polymorphism in the PDE7B promoter AM Peiro´ et al 678

herpes simplex virus thymidine kinase promoter and serves as an internal contingency table and use of the w2 statistic; Po0.05 was considered to be control to normalize for transfection efficiency. After 48 h, cells were harvested statistically significant. and assessed using a dual-luciferase reporter assay system (Promega, Madison, WI, USA). Firefly and Renilla luciferase activities were measured using a TD- RESULTS 20/20 Luminometer (Turner Design, Sunnyvale, CA, USA). The results shown were found in three independent experiments. CLL study cohort To determine whether the PDE7B promoter possesses genetic variants, Statistical analyses we sequenced samples prepared from 154 CLL patients and 93 control All determinations were performed in duplicate or triplicate. Values are subjects. This cohort represents a typical population of CLL patients expressed as mean±s.d. Comparison between groups was based on 2Â2 (that is, 62% male and 38% female subjects; mean age at diagnosis,

Table 3 Genetic variation (cSNPs) in human PDE isoforms, contigs that contain the SNPs and disorder/phenotype/disease associations of the variants

PDE Total cSNPs Contig Disorder/phenotype/disease

1A 2045 0 NT_005403.16 Major depression: association with remission produced by antidepressants drugs34 Depression: no association with treatment response33 or to citalopram35 Autism: no significant association49 1B 212 7 NT_029419.11 None 1C 1574 11 NT_007819.16 Catecholaminergic polymorphic ventricular tachycardia: association50 2A 651 12 NT_033927.7 None 3A 1963 6 NT_009714.16 None 3B 652 8 NT_009237.17 Type 2 diabetes mellitus: no association in Japanese subjects51 4A 126 14 NT_011295.10 None 4B 276 11 NT_032977.8 Associated with female-specific protection against schizophrenia19 Associated with schizophrenia and bipolar disorder17 also in Japanese population18 Not associated with risk for schizophrenia52 Associated with strokes subtypes, intracranial large artery atherosclerosis and small artery occlusion and with stroke risk factors such as diabetes and smoking53 Associated with control of prostate smooth muscle54 4C 248 10 NT_011295.10 None 4D 5442 5 NT_006713.14 Associated with greater risk of ischemic stroke20,53,55–58 in hypertensive patients59 and also in Korean,60 Chinese Han61 and Moscow62 populations Not associated with greater risk of ischemic stroke,20,63–65 including in a Sardinian population66 Associated with chronic obstructive pulmonary disease67 Associated with sleep and circadian phenotypes68 and a subtype of neuroticism69,70 Associated with chronic kidney disease in low risk subjects71 Associated with carotid atherosclerosis72 Associated with basal metabolic index and asthma73 5A 892 5 NT_016354.18 Heart rate and blood pressure: not associated with sildenafil response in men with erectile dysfunction36,74 Associated with pulmonary hypertension in patients with advanced liver disease75 Associated with progression of childhood immunoglobulin A nephropathy76 6A 464 14 NT_029289.10 Associated with retinitis pigmentosa21,77 also in consanguineous Pakistani families78 6B 294 7 NT_037622.5 Associated with congenital stationary night blindness:79 associated with retinitis pigmentosa80,81 and Usher syndrome82 6C 443 15 NT_030059.12 Not associated with retinitis pigmentosa83 7A 253 3 NT_008183.18 None 7B 1616 2 NT_025741.14 Associated with fetal hemoglobin levels in sickle cell anemia84 Chronic lymphocytic leukemia: no significant association (current study) 8A 971 5 NT_010274.16 Not associated with polycystic ovary syndrome and androgen levels in women85 8B 1196 7 NT_006713.14 Associated with serum TSH levels and thyroid function86 and with subclinical hypothyroidism in pregnancy87 9A 1072 8 NT_030188.4 Associated with susceptibility to major depressive disorder and antidepressant treatment response34 Depression: no association with treatment response49 10A 2267 11 NT_007422.13 Associated with serum TSH levels and thyroid function88 11A 2051 7 NT_005403.16 Associated with susceptibility to major depressive disorder and antidepressant treatment response34 Not associated with treatment or citalopram response in depression49 Associated with predisposition to adrenocortical tumors88 Associated with risk of familial and bilateral testicular germ cell tumours89 and marginally contributory to the development of somatotropinomas in a subset of acromegalic patients90 Associated with asthma91

Abbreviations: cSNPs, coding single-nucleotide polymorphisms; PDE, phosphodiesterase; SNP, single-nucleotide polymorphism; TSH, thyrotrophin-stimulating hormone. Contigs of PDE family members, their total number of SNPs and cSNPs listed on the National Center for Biotechnology Information dbSNP website (http://www.ncbi.nlm.nih.gov/SNP/) are shown.

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62 years). We also evaluated patient samples for expression of diseases while other data implicate associations between PDE poly- the immunoglobulin heavy chain variable mutation and 70-kDa morphisms and certain central nervous system, metabolic, cardio- zeta-associated protein, both of which are prognostic markers in vascular or neoplastic disorders. Inter-subject variability in response to CLL.26,27 (Table 1). PDE-interacting drugs associates both positively and negatively with genetic variations in PDEs. For example, genetic variations in PDE1A, Identification of a variant in PDE7B and comparison of its PDE9A and PDE11A associate with the response to antidepressants frequency in control and CLL subjects but apparently only in certain populations.33–35 Polymorphisms in In initial studies designed to identify genetic variants in PDE7B, we PDE5A appear unable to explain differences in cardiovascular effects sequenced B1.3 kb of its coding region in 43 CLL patients and in subjects who are administered sildenafil or other PDE5 inhibitors, 7 normal subjects and found limited genetic variation in this region: implicating other mechanisms besides such genetic variation as a SNP in the 5¢ upstream region, À347C4T, occurred with B2% contributing to variable responses.33,34,36 frequency (1.9% in CLL patients 6/308 alleles (all heterozygotes) and Could our results that fail to show an association of genetic variants 2.7% in 5/186, (3 heterozygotes and 1 homozygote) in controls). Thus, for PDE7B in CLL be falsely negative? Might copy number variation the overall SNP frequency was 2.2% (11/494 alleles). Subgroup explain the prominent increase in gene expression of PDE7B in CLL analysis of CLL patients classified as having either aggressive or patients?16,37 Copy number variation (1000 bp segments of genomic indolent CLL28,29 revealed a comparable frequency of SNP expression DNA with inter-individual variability in copy number38,39) has been in the two groups of patients (data not shown). Males had a slightly associated with numerous diseases.40–42 Copy number variations may higher, statistically nonsignificant (P40.1) SNP frequency (2.6%; affect gene expression43 and thus might increase expression of PDE7B 9/346) than did females (1.4%; 2/148; Table 2). Expression of the transcripts in patients with CLL. An additional mechanism for the SNP was not associated with other CLL markers (Table 1). increase in PDE7B expression in CLL could be non-coding RNAs, Use of the TFSEARCH program (http://www.cbrc.jp/research/db/ including miRNAs, expression of certain of which is increased in CLL TFSEARCH.html) to predict transcription factor binding sites and may contribute to the difference in aggressive and indolent forms revealed that the PDE7B promoter containing the SNP has putative of the disease.44 Further studies will be needed to assess gene structure binding sites for multiple transcription factors, including the sex in CLL45 and also whether the SNP alters methylation of the PDE7B determination region Y, which encodes a sex-determining transcrip- promoter, thereby changing chromosomal structure and transcription factor of the high-mobility group box family and two members of tional activity.35,46–48 the forkhead box (FOX) family, FOXA2 and FOXD3.30,31 The TFSEARCH program predicted that the À347C4T SNP abolishes CONFLICT OF INTEREST the binding site for sex determination region Y and FOXA2 but not The authors declare no conflict of interest. FOXD3. Although binding of FOXD3 was not altered by the SNP, this transcription factor is down-regulated in CLL and in a mouse ACKNOWLEDGEMENTS k model of CLL through a nuclear factor- B p50:histone deacetylase 1 This work was supported by grants from the Lymphoma and Leukemia Society, 32 co-repressor complex 3. NIH and FCVI-HGUA (Fundacioń de la Comunidad Valenciana para la investigacioń en el Hospital General Universitario de Alicante) and Conselleria Functional assessment of the PDE7B promoter SNP de Sanitat Valenciana, Spain. On the basis of possible impact of the PDE7B promoter SNP on transcription, we assessed its activity using a dual luciferase assay and found that the 347C4T variant had decreased transcriptional activity compared with the wild-type construct (Po0.001; Figure 1). 1 Beavo, J. A. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Phys. Rev. 75, 725 (1995). 2 Conti, M. & Beavo, J. Biochemistry and physiology of cyclic nucleotide phosphodies- DISCUSSION terases: essential components in cyclic nucleotide signaling. Annu. Rev. Biochem. 76, Our results indicate that the coding sequence and 5¢ upstream region 481–511 (2007). of the PDE7B gene has a SNP (À347C4T) that occurs with low 3 Goodwin, J. S. & Ceuppens, J. Regulation of the immune response by prostaglandins. J. Clin. Immunol. 3, 295–315 (1983). frequency (that is, Po3%), is located in a possible putative binding 4 Moore, A. R. & Willoughby, D. A. The role of cAMP regulation in controlling inflamma- sites for sex determination region Y, FOXA2 or FOXD3 and decreases tion. Clin. Exp. Immunol. 101, 387 (1995). transcriptional activity. Thus, although relatively rare, the SNP has the 5 Osawa, H., Niiya, T., Onuma, H., Murakami, A., Ochi, M., Nishimiya, T. et al. Others systematic search for single nucleotide polymorphisms in the 5¢ flanking region of the potential to contribute to inter-individual differences in PDE7B human phosphodiesterase 3B gene: absence of evidence for major effects of identified expression. polymorphisms on susceptibility to Japanese type 2 diabetes. Mol. Genet. Metab. 79, 43–51 (2003). On the basis of information available at the dbSNP (http:// 6 Ding, B., Abe, J., Wei, H., Xu, H., Che, W., Aizawa, T. et al. A positive feedback loop of www.ncbi.nlm.nih.gov/SNP/), PDE7B is predicted to contain 1616 phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to SNPs within the 345 718-bp contig reported in Build 129. Only two of cardiomyocyte apoptosis. Proc. Natl. Acad. Sci. USA 102, 14771 (2005). 7 Erdogan, S. & Houslay, M. D. Challenge of human Jurkat T-cells with the adenylate these are coding SNPs (found in exons 8 and 12), both synonymous, cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) and the rest are found in regulatory regions and introns. There are expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants nine SNPs identified between À473 and À1756 located in the 5¢ UTR as well as down-regulating a novel PDE4A. Biochem. J. 321, 165 (1997). 8 McCahill, A., Campbell, L., McSorley, T., Sood, A., Lynch, M. J., Li, X. et al. that are deposited in dbSNP. Thus, the À347C4T SNP discovered in In cardiac myocytes, cAMP elevation triggers the down-regulation of transcripts and this study is novel, although overall, PDE7B has a relative dearth of promoter activity for cyclic AMP phosphodiesterase-4A10 (PDE4A10). Cell Signal 20, 2071–2083 (2008). potentially functional polymorphisms. Such conservation may imply 9 Verghese, M. W., McConnell, R. 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